ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 219–226, 2005
MORPHOLOGICAL AND HISTOLOGICAL ASPECTS OF 2,4-D EFFECTS
ON RAPE EXPLANTS (BRASSICA NAPUS L. CV. KANA)
CULTURED IN VITRO
HALINA ŚLESAK, MARZENA POPIELARSKA*, AND GRZEGORZ GÓRALSKI
Department of Plant Cytology and Embryology, Jagiellonian University,
ul. Grodzka 52, 31–044 Cracow, Poland
Received January 22, 2005; accepted April 12, 2005
The effect of different durations of exposure to 2,4-D on hypocotyls and cotyledons cultured in vitro was studied in
Brassica napus L. cv. Kana. Organogenesis or callogenesis depended on the duration of explant exposure to MS
medium supplemented with 2 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid). Short treatment (1 and 3 days) with 2,4-D
resulted in rhizogenesis on hypocotyls (100% and 98% of explants, respectively) and cotyledons (80% and 54% of
explants, respectively). Adventitious shoots formed sporadically, with the highest frequency (14% of explants) on
hypocotyls cultured 3 days on MS supplemented with 2,4-D and then transferred to hormone-free medium. Histological
analysis clearly indicated that the basal part of hypocotyls is involved in root formation and callus production, and
the apical part for shoots. Meristematic sites originated from groups of cells in the cortex layer (including cells of the
endoderm), but the procambium, phloem and pericycle also showed meristematic activity. The present study indicated
that the response of explants cultured on media containing 2,4-D at constant concentration depends on the duration
of explant exposure to growth regulator.
Key words: Brassica napus L., 2,4-D, regeneration, callus, histology.
INTRODUCTION
Auxin is a phytohormone involved in the control of
various aspects of growth and development in higher
plants (Rück et al., 1993). The regulated, differential
distribution of auxin influences many processes including organogenesis and meristem patterning. Because of its multiple roles, auxin displays some
characteristics of both a hormone and a morphogen
(Friml, 2003).
The auxin signal is received by plant cells and
rapidly transduced to a wide variety of responses in the
growth and development of plant organs. These include
changes in the direction of growth, shoot and root
branching, and vascular differentiation (Leyser, 2001).
According to Petrášek et al. (2002), the division
and growth of most types of plant cells cultured in vitro
require an external source of auxin. In cultures, the
ratio of external to internal auxin concentrations is
essential for regulation of the phases of the standard
growth cycle. The type of auxin used in the medium
influences culture morphology (Hofmann et al., 2004).
Synthetic auxins such as 2,4-dichlorophenoxyacetic
acid (2,4-D) are essential constituents of culture media.
They are used in cereals, for example, for callus induction and maintenance (Pellegrineschi et al., 2004) or
for induction of somatic embryogenesis if applied at a
higher concentration (Vikrant and Raskid, 2003).
Brassica napus (oilseed rape) has been the object
of extensive tissue culture studies. Economically,
B. napus is among the most important oil crops both
for food and for industrial purposes. Many of the
Brassica napus varieties, especially those with superior properties such as high yield, disease resistance
and high quality, are fundamental material in breeding
programs (Hu et al., 1999). Some authors have described the effect of plant growth regulators on culture
of Brassica species (Rogozińska and Drozdowska, 1980;
Klimaszewska and Keller, 1985; Bagniewska-Zadworna, 2001), but precise data on the effect of the duration
of exposure to the phytohormone 2,4-D are scarce. It is
known that plant regeneration capacity may vary significantly between species, as well as between genotypes within species (Jourdan and Earle, 1989).
*e-mail: [email protected]
PL ISSN 0001-5296
© Polish Academy of Sciences, Cracow 2005
220
Ślesak et al.
The aim of the current study was to examine the
effect of different durations of exposure to a constant
2,4-D concentration (2 mg/l) on hypocotyl and cotyledon
explants of Brassica napus cv. Kana. This Polish cultivar has important properties such as frost resistance
and high yield, but no data about its growth characteristics in culture in vitro are broadly available. The
concentration of 2,4-D was chosen on the basis of previous reports indicating that 2 mg/l 2,4-D is optimal for
callus induction in Brassica (Khan et al., 2002) and in
Zea (Bronsema et al., 1997; Emons et al., 1993).
MATERIALS AND METHODS
TABLE 1. Brassica napus L. cv. Kana. Scheme of variants of
experiment cultured on induction medium (IM) and hormonefree growth medium (GM)
Variant*
No. of days on IM
No. of days on GM
control
1D
3D
5D
7D
14D
21D
28D
0
1
3
5
7
14
21
28
28
27
25
23
21
14
7
0
*variants indicated by the number of days of exposure to auxin and the
letter "D"
PLANT MATERIAL
Seeds of Brassica napus L. cv. Kana (Hodowla Roślin
Strzelce, Inc., Poland) were sterilized by soaking in
70% ethanol for 60 sec and in Ace commercial bleach
(diluted 1:1 with distilled water) for 12 min, followed
by three rinses with sterile distilled water. The seeds
were then placed in 10 cm Petri dishes on moistened
sterile filter paper and incubated at 25 ± 3˚C in the
light. Fragments of hypocotyls (~5 mm in length) and
cotyledons isolated from 5-day-old seedlings were used
as explants.
CULTURE CONDITIONS
MS medium (Murashige and Skoog, 1962) was the
basal medium. The media were solidified with 0.7%
Difco BactoAgar and adjusted to pH 5.7–5.8 with 0.1 N
HCl or NaOH prior to autoclaving at 121˚C and 1.05
bar for 21 min. Explants were cultured on induction
medium (IM) supplemented with 2 mg/l 2,4-D for 1, 3,
5, 7, 14, 21 or 28 days, and then transferred to hormonefree medium (GM) for the remaining days left, if any,
in a total 28 days of culture (Tab. 1). All cultures were
grown under a 16 h photoperiod (cool-white fluorescent
tubes 60–90 µmol photons m-2s-1) at 25 ± 3˚C.
Five explants (hypocotyls or cotyledons) per Petri
dish were inoculated, with at least three replicates per
treatment variant. Culture efficiency was calculated
from 15–35 explants in every variant. The treatments
are called 1D for one-day exposure to 2,4-D, 2D for two
days of exposure, etc.
HISTOLOGICAL ANALYSIS
The material for sectioning was embedded in Technovit
7100 (2-hydroxyethyl-metacrylate) (Heraeus Kulzer).
The cultured fragments of hypocotyls and cotyledons of
all treatment variants were sampled after 1, 3, 5, 7, and
10 or 19 days of culture and fixed in glutaraldehyde for
24 h, then washed four times in phosphate buffer
(PBS), dehydrated in a graded ethanol series for 15 min
at each concentration (10%, 30%, 50%, 70%, 96%) and
kept overnight in absolute ethanol. The next day the
samples were infiltrated in mixtures of absolute ethanol and Technovit for 1 h at each proportion (3:1, 1:1,
1:3 v/v) and stored for 12 h in pure Technovit. The resin
was polymerized with the addition of hardener. The
material was sectioned at 5 µm with a rotary microtome
(Microm, Adamas Instrumenten), stained with toluidine blue, and mounted in Entellan (Merck).
Photographs of in vitro cultures were taken under
a Zeiss Stemi SV 11 stereomicroscope equipped with
an MC80 microphotographic attachment on Kodak
film. Microscope sections were photographed with a
Zeiss Axio Cam MRe digital camera using Zeiss Axio
Vision 3.0 software.
RESULTS
MORPHOLOGICAL OBSERVATIONS
Callus induction
Callus formation was restricted to the cut edges of
explants or else started from them. Two morphological
types of callus were noted after 28 days of culture:
yellowish green, or yellow, granular callus, or white,
watery, friable callus, both on hypocotyls and cotyledons. Longer time of exposure to 2,4-D (14D, 21D or
28D variants) influenced callus structure and resulted
in the formation of yellow, compact callus (Fig. 4).
Hypocotyls. After 1 day of exposure to 2,4-D, differences in response between the apical and basal
regions of the hypocotyl were distinct. After the next
few days, epidermis sticking out from explants near
basal cut edges was observed prior to callus formation
(Fig. 1). Culture on 2,4-D-supplemented medium for
1–14 days resulted in the most abundant callus production. On explants cultured on hormone-free medium
(control), callus was of very poor quantity, but callogenesis occurred on all explants (Tab. 2).
Cotyledons. Callus was induced on the 1D explants at ~54% efficiency. Longer periods of 2,4-D
Studies on rape explants cultured in vitro
221
Figs. 1, 2. Hypocotyls of 5D variant after 5 (Fig. 1) and 7 (Fig. 2) days of culture, showing differences between the apical (a)
and basal (b) regions; intensive callus and trichome formation on the basal region, and shoot regeneration on the apical region
(Fig. 2). Bars = 2 mm. Fig. 3. Hypocotyl of 3D variant after 10 days of culture, showing short roots with long root hairs and
trichomes visible on part of the basal region. Bar = 0.5 mm. Fig. 4. Basal region of hypocotyl of 14D variant after 28 days of
culture, showing abundant callus formation with nodular structures (arrowheads), very few trichomes, and no roots. Bar = 2 mm.
exposure induced callogenesis on 88.7–100% of the
cotyledons, but on 28D explants the callus was of poor
quantity. On control explants, callus formed sporadically (8% of explants) (Tab. 3).
Morphogenic response of explants
Cotyledon explants in the control, 1D and 3D variants
enlarged significantly (~10-fold increase), and 2–4-fold
in the other treatment.
Root formation was observed on hypocotyls and
cotyledons, but shoots developed only on hypocotyls
(Tabs. 2, 3). Most frequently, regeneration was obtained indirectly via callus. The duration of exposure
to 2,4-D strongly affected root formation. Short treatment with 2,4-D (1D, 3D) was optimal for rhizogenesis;
100% of the 1D hypocotyls and 98% of the 3D variant
developed roots (Tab. 2). Similarly, rhizogenesis on
cotyledons was highly efficient in the 1D (80%) and 3D
(54%) treatments (Tab. 3).
Root hairs and trichomes observed on 1D and 3D
variants were numerous, measuring ~5 mm in length
(Fig. 3). Longer exposure of explants (14D, 21D, 28D)
resulted in inhibition of rhizogenesis on hypocotyls
(Fig. 4, Tab. 2) and cotyledons (Tab. 3).
When explants were cultured on hormone-free
medium (control), the frequency of explants producing
roots varied depending on the explant type, reaching
80% for hypocotyls and 24% for cotyledons.
Adventitious shoot formation was lower than rhizogenesis. Shoots were observed exclusively on hypocotyls (Fig. 2). The best responses were from 3D
variants (14% of explants formed shoots) (Tab. 2).
Long exposure to 2,4-D (28D) inhibited organogenesis. One shoot was formed on one hypocotyl; the rest
of the explants, both cotyledons and hypocotyls, did not
develop shoots or roots. Shoots and roots usually
formed 3–4 days after transfer to GM medium.
Observations of organogenesis and callus formation indicated that the basal region of the hypocotyl is
222
Ślesak et al.
TABLE 2. Brassica napus L. cv. Kana. Influence of 2,4-D on
morphogenetic response of hypocotyls cultured 28 days (%)
TABLE 3. Brassica napus L. cv. Kana. Influence of 2,4-D on
morphogenetic response of cotyledons cultured 28 days (%)
Variant
No. of
explants
No. of
explants
with callus
No. of
explants
with shoots
No. of
explants
with roots
Variant
No. of
explants
No. of
explants
with callus
No. of
explants
with shoots
No. of
explants
with roots
Control
1D
3D
5D
7D
14D
21D
28D
50
35
50
50
75
70
50
100
50 (100) +
35 (100) +++
50 (100) +++
49 (98.0) ++
69 (92.0) +++
70 (100) +++
50 (100) ++
97 (97.0) ++
1 (2.0)
1 (2.8)
7 (14.0)
2 (4)
1 (1.3)
2 (2.8)
0
1 (1.0)
40 (80.0)
35 (100)
49 (98.0)
11 (22.0)
16 (21.3)
0
0
0
Control
1D
3D
5D
7D
14D
21D
28D
50
35
50
40
70
80
85
115
4 (8.0) +
19 (54.2) ++
47 (94.0) ++
40 (100) ++
70 (100) +++
71 (88.75) +++
85 (100) ++
108 (93.9) +
0
0
0
0
0
0
0
0
12 (24.0)
28 (80.0)
27 (54.0)
17 (42.5)
1 (1.4)
0
0
0
Quantity of callus: + poor; ++ medium; +++ abundant
Quantity of callus: + poor; + + medium; + + + abundant
more suitable for callus and root formation; the apical
region is able to form shoots.
Changes in cell organization inside the stele
were observed on hypocotyls of the 3D variant after
5 (Fig. 7), 7 (Fig. 10) and 10 (Fig. 13) days of culture.
Sections of younger explants revealed vascular bundles
with phloem, procambium and xylem clearly visible.
Proliferation of cells started between vascular bundles
(next to phloem and procambium), but cells divided also
in the cortex layer (Fig. 7). A few days later, vascular
bundle elements were poorly recognizable (Fig. 10).
Bulbs containing meristematic cells were located above
remnants of vascular bundles. Cells of the cortex layer
were weakly attached above the proliferation area. The
growth of meristematic centers could result in callus
formation (Fig. 11) or direct rhizogenesis (Fig. 13).
Shoots with a visible root pole formed on the apical
region of the hypocotyl (Fig. 14).
HISTOLOGICAL STUDIES
A difference in response between the two regions of the
hypocotyl explants was observed after only 1 day of
exposure to 2,4-D: the basal region was more swollen than
the apical region. Sections of the 1D variant revealed
enlarged cells in the cortex layer and stele as compared
with control material cultured 1 day on hormone-free
medium (Figs. 5, 6). After 5 days, the epidermis of the 3D
variant protruded from the explants near basal cut edges.
Cell proliferation was noted in the stele, especially around
the vascular bundles and in the inner cortex (cells of
endodermis) (Fig. 7). Cortex cells were weakly attached
each other, and tissue was outgrown from the explant
(Fig. 8); this was more clearly visible in sections after
the following days of culture (Fig. 12). Parenchyma cells
were large and vacuolated, while the cells of meristematic
sites were small, with dense cytoplasm and without intercellular spaces (Fig. 9). In the region of the stele, many
meristematic sites were noted (Fig. 10). Their proliferation resulted in callus induction (Figs. 11, 12) or rhizogenesis (Fig. 13). Some unorganized xylem elements (e.g.,
vessels with scalariform wall thickening) were observed
in the calli masses.
DISCUSSION
Intra-varietal variability of the organogenetic and callogenic response to plant growth regulators may be as
great as the variability between Brassica species (Dietert et al., 1982), so the results obtained in one variety
cannot be extended to the species. In our experiments
we observed root formation only on explants cultured
on hormone-free medium or briefly exposed to 2,4-D for
Figs. 5, 6. Longitudinal sections of basal region of hypocotyl. Hypocotyl of control (Fig. 5) and 1D variant (Fig. 6) after 1 day
of culture, showing enlarged cells, especially in the stele region (asterisk). Bars = 100 µm. Fig. 7. Transversal section of
hypocotyl of 3D variant after 5 days of culture, showing induction of cell division (arrowheads) at sites between vascular
bundles (cam – procambium; ph – phloem; x – xylem). Bar = 50 µm. Fig. 8. Longitudinal section of basal region of hypocotyl
of 3D variant after 5 days of culture, showing parenchyma cells of cortex layer weakly attached to each other and trichomes
(asterisk) at end of vascular bundle. Bar = 200 µm. Figs. 9, 10. Transversal sections of hypocotyls of 3D variant after 5 days
(Fig. 9) and 7 days (Fig. 10) of culture, showing meristematic sites (arrowheads) above remnants of vascular bundles (ph –
phloem; x – xylem in Fig. 10). Bars = 50 µm in Fig. 9 and 100 µm in Fig. 10. Figs. 11, 12. Longitudinal sections of hypocotyls
of 3D variant after 7 days of culture, showing meristematic sites (arrowheads) above stele, mass of callus tissue (mc) growing
out of cortex and epidermis (ep), and organogenesis (asterisk). Bars = 200 µm in Fig. 11 and 0.5 mm in Fig. 12. Fig. 13.
Transversal section of hypocotyl of 3D variant after 10 days of culture, showing direct formation of roots. Bar = 200 µm. Fig. 14.
Longitudinal section of hypocotyl of 3D variant after 19 days of culture showing direct regeneration of shoot from apical region,
and root pole (rp) below leafy structures. Bar = 0.5 mm.
Studies on rape explants cultured in vitro
223
224
Ślesak et al.
up to 7 days. Callus formation was highly stimulated
by exposure to 2,4-D, but long exposure resulted in poor
callus growth. Shoot formation was observed exclusively on hypocotyl explants.
Callus initially formed mainly along the cut edges
of cotyledons and hypocotyls, but the cell proliferation
reached deeper parts of the explant in the following
days. Similarly, Ullah et al. (2004) reported that on
Brassica napus cv. Rainbow explants, callus proliferation started from the cut ends of the hypocotyl. Hofmann et al. (2004) reported different results in
soybean: 2,4-D induced callus production over the entire surface of the cotyledon. The type and quantity of
callus and callogenesis efficiency depended on the duration of exposure to 2,4-D and on the type of explant.
In our experiments the highest quantity of callus was
formed on hypocotyls and cotyledons after short induction on IM. Longer exposure to auxin caused poor
formation of compact calli, with many necrotic sites. No
roots and only one shoot formed on explants after
28-day exposure to auxin. Bogunia and Przywara
(2000) obtained similar results in Brassica napus cv.
Evita: media with 2,4-D as the sole growth regulator
influenced callus proliferation but no regeneration; on
hormone-free media, callus formed very sporadically.
In other Brassica species, 2,4-D was shown to be
necessary for callus formation on young hypocotyl explants (Dietert et al., 1982).
Our data confirmed previous results on Brassica
napus var. oleifera cv. Skrzeszowicki (Rogozińska and
Drozdowska, 1980), concerning the effect of 2,4-D on
callus growth, and its inhibitory effect on root formation. Similarly, in Daucus carota (Jimenez and Bangerth, 2001) the presence of 2,4-D in the culture
medium promoted callus induction and proliferation,
while culture in the absence of 2,4-D stimulated root
development at one end of the hypocotyl segments.
In our studies, shoot formation was observed,
but only on hypocotyls and with low efficiency, in
accordance with Khan et al.’s (2002) results. On
hypocotyl explants of Brassica napus cv. Oscar cultured on 2,4-D-supplemented media, shoot regeneration was induced sporadically. Klimaszewska and
Keller (1985) reported that continuous culture on
media supplemented with 2,4-D did not produce any
shoot formation. The differences between their results
and ours may be attributable to differences between
genotypes, but it is also possible that transferring
hypocotyls to hormone-free medium is critical to shoot
formation. Results obtained by Zheng and Konzak
(1999) in Triticum aestivum clearly showed that the
continuous presence of a 2,4-D concentration that satisfied callus induction inhibited further development
and subsequent plant regeneration. Switching the culture to a lower 2,4-D concentration in the medium is
needed for efficient regeneration of plants from calli.
The continuous presence of 2–4 mg/l 2,4-D during
induction beyond the critical point at which genes
encoding products for plant regeneration are expressed
is detrimental to the normal development of calli and
may cause the loss of their regeneration capacity. In
other species, shoot formation was observed when 2,4D was accompanied by other growth regulators. Such
observations were described in, for example, Pleione
formosana: medium with 2,4-D induced callus only,
while combining 2,4-D with TDZ (thidiazuron) produced shoot buds (Lu, 2004). Sairam et al. (2003)
demonstrated callus induction and shoot bud differentiation from excised ends of soybean cotyledons after
culture on media containing 2,4-D and benzyladenine.
Many studies have confirmed that the use of 2,4-D
is critical to induction of somatic embryogenesis (e.g.,
Choi et al., 1998; Wang and Wei, 2004). However, in
some species and genotypes, continuous long-term
auxin treatment can inhibit somatic embryogenesis
completely (Anzidei et al., 2000). The 2,4-D concentration used in our experiment (2 mg/l) was optimal for
induction of embryogenic callus derived from immature embryos of wheat (Zhang et al., 2000). In contrast,
the same concentration of 2,4-D in medium for culture
of maize callus yielded only rhizogenic callus and root
formation (Emons et al., 1993). In the present experiment, no somatic embryo formation was obtained.
Since induction of somatic embryogenesis requires
long-term culture – for example, 2, 4 and 6 months in
callus of Foeniculum vulgare (Anzidei et al., 2000) – 28
days of culture may be too short for somatic embryos
to develop on B. napus explants.
Culture with 3 and 5 days of exposure to 2,4-D
induced the most shoots (14% and 4%, respectively) and
relatively high root production on hypocotyls (98.0%
and 22.0%, respectively). The low efficiency of shoot
induction on cultured tissue in the present experiment
may be connected with the reaction of Brassica napus
cv. Kana to the composition of the culture medium. It
is known that culture conditions as well as the plant
genotype have a significant impact on shoot regeneration frequency (Hu et al., 1999). In Brassica napus ssp.
oleifera cv. Westar, shoot organogenesis occurred only
in the presence of NAA in the culture medium (Klimaszewska and Keller, 1985), and on Brassica napus cv.
Oscar explants the highest frequency of shoot regeneration was achieved on medium with 2 mg/l BAP and
0.5 mg/l IAA (Khan et al., 2002).
Histological observations revealed that cell proliferation started between and above the vascular
bundles of hypocotyls. Meristematic sites seemed to
originate from cells of the pericycle or endodermis. It is
well known that the pericycle of roots has meristematic
potential. Morphological observations showed that root
formation was more efficient on the basal region of the
hypocotyl. In this part of the explant, the pericycle
could more easily switch to the organogenesis pathway.
Similar cell divisions near vascular bundles leading to
Studies on rape explants cultured in vitro
the formation of meristematic centers were observed in
Gentiana pneumonanthe leaf explants (Bach and Pawłowska, 2003).
Our observations indicated that the response of
hypocotyl explants to hormone-free media and to media
supplemented with 2,4-D is highly polarized. Root,
trichome and callus formation started mainly from the
basal region of the hypocotyl, but the few shoots obtained in the experiment appeared only on the apical
region of the hypocotyl. Similar observations were
made in bean (Angelini and Allavena, 1989). This indicates that auxin significantly increases the competition
response of the two hypocotyl regions.
Plant explants in culture are usually inoculated on
media containing stable concentrations of growth regulators, and then are moved to new media after a few
weeks. The present study confirmed that the response
of explants cultured on media containing 2,4-D depends not only on the concentration of the phytohormone but also, and to a great extent, on the duration
of exposure to growth regulator. The meristematic sites
probably could develop in roots or shoots only after
transfer to hormone-free medium. Continuous longterm auxin treatment inhibited the morphogenic response of the explants, a phenomenon which should be
considered when experiments are planned.
ACKNOWLEDGEMENTS
The authors wish to express their deep sorrow at the
passing of Professor Lesław Przywara. Professor Przywara was our mentor and the supervisor of our doctoral
theses. He introduced us to the fascinating field of plant
experimental embryology and tissue culture.
It is a great honor for us to participate in this
special volume dedicated to the memory of our teacher,
Professor Lesław Przywara.
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